Familial Mediterranean fever (FMF) is an inherited disorder of inflammation characterized by episodes of fever with abdominal pain, pleurisy, and/or arthritis; some patients also develop systemic amyloidosis, leading eventually to renal failure. FMF is caused by recessive mutations at a locus designated MEFV that our laboratory mapped to chromosome 16p13.3 in 1992. Our objective since that time has been to identify the causative gene and its mutations by positional cloning. By the beginning of this year we had narrowed the candidate region to 240 kb, and had assembled a transcript map from this interval. During the last year we continued to identify and characterize transcripts from this interval. Partial transcripts that had already been identified were extended, using conventional library screening, solution hybridization, and rapid amplification of cDNA ends. In parallel, a region of approximately 90 kb in the center of the candidate interval underwent finished sequencing in order to find potential coding regions and to define new polymorphic markers. As a direct result, 3 new DNA polymorphisms, D16S3403, D16S3404, and D16S3405, were identified, permitting a further narrowing of the candidate interval to approximately 115 kb. Mutational analysis demonstrated that one of the genes encoded within this 115 kb, clone v75-1, is likely to be the gene causing FMF. This clone was isolated by solution hybridization of a leukocyte cDNA library with biotinylated oligonucleotide probes derived from two trapped exons. Three different missense mutations were identified in affected individuals, but not in a panel of almost 300 normal chromosomes. The three missense mutations are clustered within 46 amino acids of one another, near the carboxy terminus of the protein. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory processes.